Generating recombinant affinity reagents with arrayed targets

ABSTRACT

Methods for screening of affinity reagents for many target proteins of interest simultaneously. Arrayed targets (e.g., peptide, protein, RNA, cell, etc.) are used in affinity selection experiments to reduce the amount of target needed and to improve the throughput of discovering recombinant affinity reagents to a large collection of targets.

CROSS REFERENCE

This application claims priority to U.S. provisional patent application62/218,362 filed on Sep. 14, 2015, which is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

Embodiments herein relate generally to the field of high-throughputscreening of affinity reagents for many targets of interestsimultaneously.

BACKGROUND OF THE INVENTION

Affinity selection is a process that utilizes so-called “displaytechnologies” (ribosome-, mRNA-, and phage-display) to isolaterecombinant affinity reagents for a given target (e.g., peptide,protein, RNA, cell, etc.). One of the bottlenecks of this process isproducing the targets that are needed for the affinity selection.

In fact, many affinity reagent pipelines devote a significant amount ofresources to generating high-quality target proteins. In addition,because the targets used in selection are sometimes labile (i.e., theyare prone to degradation and denaturation), affinity selections fail.

Consequently, achieving a high-throughput and efficient affinityselection process remains problematic.

SUMMARY OF THE INVENTION

Embodiments herein relate to cost-effective screening of affinityreagents for many target proteins of interest simultaneously.Consequently, affinity reagents can be discovered that will potentiallyaid in detecting, inhibiting, or activating target proteins.

In various embodiments, arrayed targets (e.g., peptide, protein, RNA,cell, etc.) are used in affinity selection experiments to reduce theamount of target needed and to improve the throughput of discoveringrecombinant affinity reagents to a large collection of targets.

Preferably, protein-target method embodiments herein use arrayedmaterial that is translated shortly before each round of selection, asusing labile protein targets is found to be less effective than usingfreshly made target samples.

These and other aspects of the embodiments disclosed herein will beapparent upon reference to the following disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Affinity selection via phage-display using arrayed targets.First, the naïve library is incubated with freshly-translated target onthe array. Next a wash step removes non-binding clones from the array,while binding clones are retained. Phage particles remaining on thearray are then eluted from their respective targets and are amplifiedfor subsequent rounds.

FIG. 2. Proposed pipeline for identifying recombinant affinity reagents.

FIG. 3. Detecting phage particles on NAPPA array. (A) Schematic of arraylayout containing 13 unique targets. (B) Detection of phage particles(1:1000) displaying a known MAP2K5 binder. (C) Detection offreshly-translated protein in wells via the halo epitope.

FIG. 4. Phage library affinity selection of the protein targets MAP2K5,CTBP1, SARA1A, and CDK2. Phage Input is normalized by counting coloniesof TG1 cells infected by each library. Approximately,R1-lib:R2-Lib:Naïve Lib=1:1:10.

FIG. 5. Graphs of colony counts for the naive library, Round 1, andRound 2 of selection for the protein targets of the experiment in FIG.4.

FIG. 6. RPS6KA3 is an antigen that is not selected in the initialenrichment experiment. Therefore, we would not expect increased signalafter two rounds of enrichment. Consistently, this data shows RPS6KA3was negatively selected.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments herein relate to arrayed targets that are used in affinityselection of display libraries. Traditionally, affinity selectionprocedures use individual protein or peptide as targets, which have alow throughput (i.e., one at a time) and require a significant amount oftarget.

With the advent of so-called “array” technologies, one is able to a)spot proteins or peptides on an array or b) synthesize in situ thousandsof fresh target proteins on a solid surface (array) within a few hours.Thus, one solution to the low-throughput/large amount of targetlimitations is to affinity select with arrayed targets. For example,synthetic peptides or proteins can be spotted or captured in arrays onglass slides. Alternatively, proteins can be synthesized in situ inindividual spots of an array. The method of choice of in vitro synthesisand capture of proteins in spots is the nucleic-acid programmableprotein array (NAPPA).

In NAPPA, cDNAs coding for the target of interest are cloned into anexpression vector, which generates a fusion (Halo Tag, GST, etc.) to thetarget, and spotted onto an aminosilane-coated glass slide. Then to eachspot, a HeLa cell in vitro transcription-translation reagent is added,whereby the fusion gene is transcribed into mRNA and translated. Thenascent proteins are captured to the slide with an antibody/affinityagent to the fusion partner (i.e., HaloTag-ligand, α-GST antibody) thatis spotted adjacent to the DNA during the manufacture of the array. Thismethod allows for up to thousands of protein targets to be arrayed.

In the embodiments disclosed herein, targets, which have been generatedby NAPPA, are used in affinity selection experiments (FIG. 1). By usingfreshly translated protein/peptide targets (i.e., within approximately 1to 24 hours), the likelihood of denaturation of targets during storageand freeze thawing is reduced.

In addition, this process is performed on an array, which can producemany fresh protein samples. Therefore, one is able to perform amultiplexed selection on multiple targets using a single library,thereby reducing the time and cost of generating these reagents.

In some method embodiments, the process includes first performing tworounds of multiplexed panning on the array, followed by a separationround using a macrowell format (that still utilizes freshly-translatedtarget protein), which separates binding phage based on their cognatetarget (FIG. 2). Finally, the resulting clones from pools isolated inthe separation round are analyzed via macrowell analysis or ELISA toidentify clones with the highest affinity for the target.

Recently, we have shown that an M13 bacteriophage, which displays aknown binder to a particular protein target, can be detected to bind theNAPPA-generated and arrayed form of the same target (FIG. 3). A strongsignal occurs when the virions are either pure or mixed 1 to 100 with aphage library containing 2×10¹⁰ members. This finding demonstrates thatvirions displaying a binder can bind selectively and efficiently to aNAPPA-generated target protein.

EXAMPLES One Construction and Array Production

All genes of interest were cloned in pJFT7_nHALO or pJFT7_cHALO, theNAPPA compatible expression vectors. These expression vectors allow thein vitro expression of proteins of interest with a terminal HaloTag.Protein arrays were constructed through a contra capture concept asdescribed (1).

Enrichment

Array displaying MAP2K5, CTBP1, SARA1A and CDK2 were constructed andexpressed. Initial non-enrichment phage library was incubated and washedto allow binding. Mild acid (0.2M Glycine pH2.0) wash was used to removethe bond phage particles and immediately neutralized using 1M Tris-Cl(pH9.1). E. coli were then infected with the collected phage for titringand amplification as previously described (2).

Probe Libraries on Arrays

To evaluate the enrichment efficiency, same input of non-enrichedlibrary, R1 and R2 were probed on the protein microarray containingMAP2K5, CTBP1, SARA1A, CDK2 and RPS6KA3 for 1 hr at RT, followed by theM13 antibody at 1:500 dilution for another 1 hr at RT. Alexa Fluor 647or Alexa Fluor 555 conjugated anti-mouse IgG secondary antibodies(Thermo Scientific) were then incubated with the array for 1 hr. Afterproper wash, slides were scanned at 10 micron resolution using TECANscanner.

REFERENCES

-   1. Karthikeyan K, Barker K, Tang Y, Kahn P, Wiktor P, Brunner A,    Knabben V, Takulapalli B, Buckner J, Nepom G, LaBaer J, Qiu J. A    Contra Capture Protein Array Platform for Studying    Post-translationally Modified (PTM) Auto-antigenomes. Mol Cell    Proteomics. 2016 July; 15(7):2324-37. doi: 10.1074/mcp.M115.057661.    Epub 2016 May 2. PubMed PMID: 27141097; PubMed Central PMCID:    PMC4937507.-   2. Kay, B. K. et al., eds. Phage display of peptides and proteins: a    laboratory manual

The following claims are not intended to be limited to the embodimentsand other details provided herein.

1. A method for affinity selection with arrayed target material,comprising the steps of: a) incubating a display material with saidarrayed target material, b) washing said arrayed target material toremoves non-binding display material from the arrayed target material,c) eluting from the arrayed target material any binding displaymaterial, d) amplifying said binding display material; and e) performingat least a further round of affinity selection based on steps a)-c). 2.The method of claim 1, wherein said array target material comprises apeptide, protein, RNA, DNA or cell.
 3. The method of claim 1, whereinsaid display material comprises a ribosome-, mRNA-, or phage-display. 4.The method of claim 1, further comprising the step of isolating bindingdisplay material and analyzing via macrowell analysis or ELISA toidentify display material with the highest affinity for the array targetmaterial.
 5. A method for affinity selection with arrayed targetprotein, comprising the steps of: a) incubating a phage display materialwith said arrayed target protein, b) washing said arrayed target proteinto removes non-binding phage display material from the arrayed targetmaterial, c) eluting from the arrayed target protein any binding phagedisplay material, d) amplifying said binding phage display material; ande) performing at least a further round of affinity selection based onsteps a)-c).
 6. A method for negative selection of an arrayed targetmaterial, comprising the steps of: a) incubating a display material withsaid arrayed target material, b) washing said arrayed target material toremoves non-binding display material from the arrayed target material,c) eluting from the arrayed target material any binding displaymaterial, d) amplifying said binding display material; e) performing atleast a further round of affinity selection based on steps a)-c); and f)analyzing via macrowell analysis or ELISA to identify display materialwith decreasing affinity for the array target material versus a firstround of selection.
 7. The method of claim 5, wherein said displaymaterial comprises a ribosome-, mRNA-, or phage-display.
 8. The methodof claim 5, wherein said arrayed target protein comprises one or more ofMAP2K5, CTBP1, SARA1A, CDK2 and RPS6KA3.
 9. The method of claim 5,further comprising the step of isolating binding display material andanalyzing via macrowell analysis or ELISA to identify display materialwith the highest affinity for the arrayed target material.